The following viruses are examples of viruses that are propagated in embryonated eggs or in primary cultures of chicken embryo fibroblasts prepared from embryonated eggs: avian influenza virus, avian reovirus, fowlpox virus, psittacine herpesvirus (Pachecco's herpesvirus), swine influenza virus, equine influenza virus, Newcastle disease virus, falcon herpesvirus, pigeon herpesvirus, infectious bursal disease virus, infectious bronchitis virus, Marek's disease virus, turkey herpesvirus, chicken anemia virus, avian encephalomyelitis virus, pigeon pox virus, canary pox virus, quail pox virus, avian polyomavirus types I and II, and avian adenovirus types I, II, and III. Further, following are examples of human viruses that are propagated in embryonated eggs or in primary avian cell cultures: Influenza A and Influenza B viruses, West Nile virus, yellow fever virus, Chikungunya virus, Dengue fever virus, and most encephalitis viruses.
Detection of any one of the above-identified viruses as the etiological agent for a diseased animal or human can be determined by serological based assays or unequivocally by virus isolation (VI) diagnostic assays. For many of these viruses, live or killed vaccines are available to protect the animal or human from infection by the aforementioned viruses.
Current influenza vaccine production technology is largely restricted to growing viruses in embryonated chicken eggs, often requiring one to two eggs per dose [29,30]. This production scheme thus requires that hundreds of millions of “clean” embryonated eggs are available each year to meet the demand for influenza vaccines. In addition, this scheme requires significant downstream processing to purify virus away from egg components and is subject to significant loss of product if the eggs used are found to be contaminated with exogenous agents, such as Salmonella spp. From selection of vaccine viruses (to be included in any given year's vaccine) to actual production can take as long as six to nine months. Many scientists and vaccine experts have expressed concern that this is far too long in the face of a potential pandemic [21, 29, 30]. There are also concerns that individuals who are allergic to eggs may experience adverse reactions to egg-derived vaccines.
Because of safety and consistency issues with egg-derived influenza vaccines, there has recently been a push from both regulatory agencies and major vaccine manufacturers to adopt a continuous cell culture-based influenza vaccine production system (FDA, 2001). The recent emergence of potentially pandemic influenza viruses, such as high pathogenicity H5N1 strains has also highlighted deficiencies in the ability of current manufacturing systems to rapidly respond to a pandemic [21, 30]. While influenza viruses generally grow efficiently on primary chick kidney cells [11, 15], this system would be subject to many of the same concerns and issues surrounding egg-derived vaccines, particularly the potential presence of harmful contaminating pathogens.
Thus, a well-characterized continuous cell line that can be used to establish a master cell bank free of exogenous pathogens is valuable. The “PBS-1” cell line is one such known cell line. PBS-1 is an immortalized chick embryo cell line that is capable of growing viruses to high titers. Viruses grown in PBS-1 cells are released into the culture fluid without the need for exogenous proteases, thus simplifying downstream processing. PBS-1 cells are free of any exogenous agents, are non-tumorigenic, and are readily adaptable to a variety of culture conditions, including growth on microcarrier beads. The PBS-1 cell line, and methods of use of this cell line, are disclosed in five U.S. Pat. Nos. 5,827,738; 5,833,980; 5,874,303; 5,866,117; and 5,989,805, all of which patents are incorporated herein as if fully set forth. Due to its embryonic nature, the PBS-1 cell line has been shown to be susceptible to a wide range of viruses.
A cell line that adapts to serum free conditions shows advantages in the vaccine production process. The high protein concentrations present in culture media enriched with serum increases the complexity of product purification. High lot-to-lot variation of serum can be found because serum is poorly defined. Additionally, another critical aspect of using serum involves a high risk of contamination by viruses, mycoplasma, and prions [5]. Washing steps and medium exchange could be reduced if serum-free conditions are applied which can also reduce the risk of contamination and increase productivity.
Currently, there are three continuous cells lines which meet regulatory requirements and have been shown to successfully replicate influenza A and B viruses. These cells are Madin-Darby canine kidney (MDCK) cells [28], African green monkey kidney cells (Vero) [14], and human fetal retinoblast cells (PER.C6) [19]. All three of these cell lines have been adapted to grow in serum free media [14, 19, 28].
The PBS-1 cell line requires serum or animal product (e.g., bovine serum) for growth. As such, the utility of PBS-1 for manufacturing human vaccines is limited by the potential for contaminating elements such as bovine spongiform encephalopathy (BSE), a progressive neurological disease that is fatal to cattle and has been associated with variant human Creutzfeld-Jacob syndrome. Accordingly, it is desirable to have a cell line derived from the PBS-1 cell line that is adapted for growth in serum-free or animal-product free conditions.